AN ABSTRkCT OF THE THESIS OF
for the Master of Science
Norman Lewis Baldwin
(Degree)
(Name)
in Fiseries presented on
,7
- ____
(Date)
jor)
Title:
J/
I /
STUDIES OF THE HOST-PARASIIE RELATIONSHIPS OF THE t1SALMQN
1
POISONING" TREMATOIJE--
Abstract approved
Redacted for privacy
'Dr. Raymond E. Millernann
The effect of experimental infection with the "salmon poiscning"
fluke, Nanophyetus salmincola, on five species, including three subspecies, of salmonid fish was studied.
The estimated numbers of
cercariae that killed in 24 hours 50% of kokanee (Q 2rhnchus nerka),
Montana black-spotted cutthroat trout (Salmo
arki lewisi.), ktlantic
salmon (S. salar), coho salmon (0. kisutch), rainbow trout (.
gairdneri), and coastal cutthroat trout (S. clarki clarki), all between 30 and 37 millimeters in fork length, were 58, 74, 110, 200,
295, and 430, respectively.
This range in sensitivity of the dif-
ferent species of fish to the parasite was correlated with the
natural duration of the host-parasite relationships.
The symptomatic,
gross and histopathologic changes associated with.penetration and
migration of N. salmincola cercariae in the above trout and salmon
species and in Lahontan cutthroat trout (S. clarki henshawi) are
described.
Penetration and migration of cercariae in fish were
'This study was supported by ablic Health Service Research Grant
5 ROl A106599-02, from the National Institute of JJergy and Infectious DLeases.
Parasites were found inside fin rays and blood vessels,
studied.
and were present in internal organs within 1 hour after epidermal
penetration.
Effects of different salinities on emergence, viability,
and infectivity of N. salmincola cercariae were determined.
Cercariae
emerged from the snail, Oxytrema silicula, in waters having salinities
that ranged from 2 to 20 ppt.
Cercariae survived the longest (68 to
75 hours) in waters having salinities from 2 to 6 ppt.
Cercariae
penetrated fish at all test salinities (12 to 20 ppt) except at 4
arid
ppt.
snails.
Observations were made on emergence of cercariae from
Cercariae were present in the blood of the afferent
ctenidial sinus.
The parasites passed dom the ctenidial leaflet
through a central sinus to the leaflet apex and then into the
mantle cavity.
Studies on the host-parasite relationships of the "salmon
poisoning" trematode--Nanophyetus salmincola (Chapin)
by
Norman Lewis Baldwin
A THESIS
submitted to
OREGON STATE UNIVERSITY
in partial fulfillment of
the requirements for the
degree of
Master of Science
June 1967
Redacted for privacy
Associate Professor of Fisheries
In Charge of Major
Redacted for privacy
Redacted for privacy
Dean of Graduate School
Date thesis is presented
''
rped by Virginia Hall Keflar for Norman Lewis Baldwin
ACKNOWLEDGNTS
I deeply appreciate the encouragement and guidance of Dr.
Raymond E. Millernann throughout my research and for his patient
assistance given during the preparation of this manuscript.
Gratitude is expressed to Mr. Gary A. Gebhardt and Mr. Pete
A. Irberg for their assistance given during this research work.
I gratefully acknowledge the assistance of the following
organizations and individuals in supplying the fish used in this
study:
The Oregon State C-e.me Commision, Mr. Christopher C.
Jensen,
the Commission's Fish Culture Supervisor, Mr. K. E. Morton, Mr. R. A.
Evans, and Mr. C. T. Roadarmel, the Commission's Hatchery Superin
tendents of the Wizard Falls, Kiamath, and Cedar Creek hatcheries,
respectively; the Fish Commission of Oregon, Mr. Ernest Jeffries,
the Commission's Fish Culture Director, and Mr. K. C. Bennett, the
Commission's Hatchery Superintendent of the Alsea River Salmon
Hatchery; and the Bureau of Sport Fisheries and Wildlife,
Division
of Fish Hatcheries, Mr. Marvin Smith, the Division's Regional Super
visor, and Mr. Richard Bigej, Hatchery Biologist, U. S.
National
Fish Hatchery, Creston, Montana.
I am especially indebted to my wife, Donna, for encouragement
and understanding as well as for her typing of the rough draft.
TABLE OF CONTENTS
PAGE
I NTRODtJCTION.
MATERIA.IIS
I.
AND I4ETHODS ......
.
.
.
.
.
.
.
1
.
7
Effects of N. salinincola cercariae on salmonid
fishes under experimental conditions .......
7
........... .
7
Snai1culture...
................. 8
Fish
Collection of cercariae ............
9
Infection of fish--mortality studies ...... 10
Penetration and migration of cercariae in fish:
histopathology.
II.
III.
.
.
.
.
.
.
.
.
.
13
........
13
.
1.
Percutaneous exposure.
2.
Alimentary canal exposure.
.
I.
.
.
.
13
snails held in waters of different salinities.
.
14
16
.
.
.
Thiergence of N. salmincola cercariae from
Effect of different salinities on viability and
.
.
.
.
Viability of cercariae. ........ .
.
.
.16
Infectivity of cercariae.
.
.
.
.
.18
.
.
.
.
.
.
.
.
17
aergence of N. salmincola cercariae from the
snail, 0. silicula.
RE5 ULTS .
.
.
.
infectivity of N. salmincola cercariae
IV.
.
......... .
.
.
.
.
.
. ..... .
.
.
.
.
.
.
.
.
.
...... .
19
Effects of-N. sa]iniricola cercariae on salmonid
fishes under experimental conditions
.
.
.
.
.
.
19
Infection of fish--mortality studies ...... 19
Penetration and migration of cercariae
in fish .................. .
.
. 25
1. Percutaneous exposure .......... .
a.
Symptomatic arid pathologic changes.
25
. 30
2. Alimentary canal exposure ......... 33
II.
nergence of N. saimnincola cercariae from snails
held in waters of different salinities ....... 34
III. Effect of different salinities on viability and
infectivity of N. salmincolacercariae .......
34
Viability of cercariae ............. 34
Infectivity of cercariae ............. 36
IV.
Emergence of N. sa]mincola from the snails,
0. silicula .
.
.
. .
. ...............
38
DISCUSSION ............................
40
BIBLIOGR4i'HY .
46
. ........................
STUDIES ON THE HOST-PARASITE RELATIONSHIPS OF THE "SALMON
POISONING" TREMATODE---NANOPHYETUS SALMINCOLA (cHAPIN)
INTRODUCTION
Evidence for the pathogenicity of the "salmon poisoning" fluke,
Nanophyetus
to fish is inconclusive.
Ward and Mueller
(1926) examined brook trout fry that had died in an Oregon hatchery
during a severe epizootic.
The fish were heavily infected with en-
cysted metacercariae of a digenetic trematode later identified as
N. salmincola.
The above authors believed that the parasites were
the cause of the losses.
They found a direct relationship between
the degree of popeye (exophthalmia) and the number of parasites in
the optic nerves.
Siinms, Donhan and Shaw (1931) and Simms et
.
(1931) believed that fish could tolerate large numbers of parasites;
e.g. they found 14,062 parasites in an apparently healthy naturally
infected cutthroat trout 4.6 inches long.
Simms (1933) reported
heavy N. salmincola infections in young rainbow and brook trout
that were dying in an Oregon hatchery.
iri many of the fish.
Exophthalmia was observed
Simms (1933) also found heavy infections in
apparently healthy fish from streams and hatcheries.
He reported
that there were no deaths or signs of infection in young rainbow
and brook trout that had heavy infections after laboratory exposure
to infected snails.
Shaw et al.
(1934) exposed young rainbow and
brook trout, approximately 2 inches long, to infected snails.
The
fish remained in good condition for periods up to 90 days even
though some of the fish were exposed for 55 days to snails and were
2
heavily parasitized.
Bennington (1951) and Bennington and Pratt
(1960) concluded, on the basis of results from laboratory experiments
in which fish and infected snails were placed together in aquaria,
that the parasite is pathogenic.
In these experiments, 12 chinook
salmon, 2 dace, 1 sculpin, and 2 goldfish died 6 to 24 hours after
exposure to infected snails.
Bennington (1951) stated that the
parasite "may be quite pathogenic to young fish" during the season.
of low water in streams having high populations of infected snails.
Farrell and Lloyd (1962) reported that rainbow trout were killed
after laboratory exposure to infected snails.
They stated that the
degree of pathogenicity was directly proportional to the rate of accumulation of cercariae by fish.
All of the above studies were con-
erned with approximately 24 hour mortalities of fish.
There is no
information on long-term mortality of fish infected with N.
salmincola..
Regarding the symptomatic, gross and histopathologic changes
associated with the infection of fish, Ward and Mueller (1926), as
noted above, believed that the parasites caused exophthalinia.
Bennington and Pratt (1960) and Farrell and Lloyd (1962) were unable to produce this condition experimentally.
Bennington and Pratt
(1960) reported that a local dermatitis developed in salmon where
cercariae had penetrated.
They also observed extensive lesions of
the fins, gills, tails, retinas, and corneas of wild infected fish.
They concluded that these effects were produced by migrating cercariae because histological sections contained numerous metacercariae
adjacent to the lesions.
They stated that the metacercariae were
3
not harmful to fish.
Wood and Yasutake (196) studied the histopath
ology associated with the metacercariae in naturally infected hatcheryraised yearling coho salmon.
They found little tissue reaction or in-.
flaxnation around the parasites.
Hyperplasia of epithelial cells or
fibrous walls were observed around some parasites.
There was eridence
of "marked obstructive and mechanical injury" to the muscle fibers of
the heart ventricle, the retina, the kidney tubules, the pancreatic
tissue, and the gall bladder wall.
Metacercariae observed in the
Wood and
aorta were considered large enough to obstruct this vessel.
Yasutake (1956) concluded that an infected fish is weakened physiologically in practically every organ.
Simms, Donham and Shaw (1931) concluded on the basis of indirect
evidence that cercariae entered the fish through the gills.
Sinitsin
(1930) stated that most of the cercariae attached to the fish's abdomen and moved along it to the urinary aperture and thus reached the
kidney by this route; however, it is not clear whether he was observing
the cercarige of N. salmincola or those of some other trematode.
Bennington and Pratt (1960) found that few cercariae crawled on the
skin or through the urinary aperture and that any part of the fish
could be penetrated, the point of entry depending on the point of contact.
Penetration took place within 30 seconds to 2 minutes; the
cercariae then rested for a short time, and then migrated into deeper
tissue.
Bennington and Pratt (1960) also reported that cercariae that
had penetrated into the tail of a fish entered blood vessels between
the rays and crawled caterpillar-like to the caudal peduncle.
They
suggested that the parasites reached the kidney via the renal portal
system.
I have developed methods for maintaining infected snails during
all months of the year, stimulating infective cercariae to emerge from
snails, exposing fish to known numbers of cercariae, and determining
the total number of encysted metacercariae in small fish.
Thus, I
have been able to study the effects of known numbers of parasites on
the fish host under controlled conditions.
uch studies have not been
done previously.
Oxytrema silicula, the snail host for N. salmincola, occurs
predominantly in the freshwater parts of streams.
Gebhardt (1966)
was the first to report the occurrence of the snail in brackish water.
He collected snails from the Alsea, Siletz, and Yaquina Rivers in
western Oregon during all seasons of the year.
The maximum and mini-
mum salinities recorded by him at the farthest points downstream in
which he found snails were 4.2 and 0.8 ppt, 4.2 and O.4 ppt, and 11.2
and 4.2 ppt in the Alsea, Siletz, and Yaquina Rivers, respectively.
Karpevich (in Zhadin, 1965) stated that molluscs can be adapted
to tolerate salinities greater than those that occur in their normal
habitat.
He studied three species of the bivalve, Dreissena.
One
species was found exclusively in freshwater, whereas, the other two
occurred in brackish water.
D. polymorpha, which is found in fresh-
water of the Volga River died when the salinity was increased to 6 or
7 ppt.
D. andrusovi and ID. casp, which are found usually in waters
with salinities from 4 to 11 ppt (Caspian Sea), could not tolerate
freshwater or waters with salinities above 17 ppt.
Published studies on emergence of cercariae from snails in waters
5
with different salinities, and on the salinity tolerance of cercariae
Rees (1948)
have been done using only cercariae from marine snails.
studied the effect of different dilutions of seawater on emergence of
Cercaria
from the marine snail Nucella
ll!.
Snails were
held for 24 hours in water having a salinity of 35 ppt and were then
placed in water having a salinity of 30 ppt for 24 hours.
At the end
of each succeeding 21-1-hour period snails were placed in waters of de-
creasing salinities at 26, 21, 17, and 13 ppt.
Cercariae stopped
emerging from snails when the salinity of the water was decreased to
17 çpt.
The snails died after 12 hours in water at 13 ppt salinity.
Stunkard and Shaw (1931) studied the effect of different dilutions of seawater and tap water on the longevity of the following
species of marine cercariae:
tle
and Cercaria
prvicaudata from the marine snail Littorina litor; Cercariaeum
lintoni, Cercaria guissetensis and Cercaria varilandis from Nassa
obsoleta; and Cercaria sensifera from Urosalpinx cinereus.
Most of
the cercariae of all species died within 1 hour in tap water.
In
one-eighth seawater, cercariae were active for consiaerable periods
of time and some of them were swimming after 4 hours.
Cercariae were
only slightly affected when held in one-fourth seawater.
Sindermann and Gibbs (1953) determined the salinity tolerance of
Microbilharzia variglandis, a parasite of the marine snail Nassa
obsoleta.
Cercariae survived salinities to 3.2 ppt using different
dilutions of seawater.
Fifty per cent of the cercariae were dead in
12 hours in water at 12 ppt salinity.
Some cercariáe survived for
more than 1 hour in distilled water.
There have been no published
6
studies on cercariae emerging from freshwater snails in brackish water.
Shaw (personal communication) in 1930 exposed N. salmincola cercariae,
obtained from crushed snails, to seawater and to various concentrations
of sodium chloride. AU cercariae in undiluted seawater died immediately.
At 20, 12.5, and 8.3 ppt, all cercariae were dead at 2, 7,
and 45 hours, respectively.
All control cercariae in tap water at
room temperature were dead at 41 hours.
This study was undertaken to determine:
(1)
the effects of
experimental infection with N. salmincola on five species, including
three subspecies, of salmonid fish with respect to both short-term
and long-term mortality, an
symptomatic, gross and histopathologic
changes associated with cercarial migration, and the routes of migration of cercariae in fish; (2) if cercariae will emerge from snails
held in waters of different salinities; (3) the effect of different
salinities on the viability and infectivity of cercariae; (4) the
route of emergence of cercariae from the snail.
7
MATERIALS AND METHODS
I.
Effects of N. salmincola cercariae on salinonid fishes under
experimental conditions.
Snail culture
Two- to three thousand snails (a. silicula) were collected from
the Big Elk River, 3.5 miles east of Elk City, Oregon, between July 7,
1965 and December 1, 1965.
The mean maximum diameter of the shell
aperture of 263 of these snails was 6.7 millimeters and the range
was 6.6 to 10.8 millimeters.
Snails were kept in plastic trays 4 feet
long, 2 feet wide, 6 inches deep, containing chlorine-free tapwater
at 17 to 18°C.
The trays were subdivided into nine compartments each
containing 70 snails and each provided with a continuous supply of
air.
Fresh water was added to the trays weekly to compensate for
evaporation.
The snails were fed dried alder leaves previously leach-
ed in hot tapwater for 8 hours, and chicken mash pellets consisting of
ground mixed cereal grains.
Leaves were added weekly and the pellets
were sprinkled lightly into each compartment every 3 to 4 days.
Broken clam shells and sand were added to provide a source of calcium
and other minerals.
Under the above conditions, most of the snails infected with
i.
salmincola were maintained for 1 year from the date of collection.
The degree of infection in the snails did not decline appreciably,
and mature cercariae were obtained during all months of the year.
Infected and noninfected snails laid egg masses from December 15, 1965
to February 1, 1966 and from March 1, 1966 to August 1966.
Some of
these eggs were saved and kept In jars containing aerated tapwater
at 17 to 18°C.
The eggs hatched approximately 10 days later and these
snails lived for 6 months.
Fish procedures
Fish representing five species of salmonicis, 29 to
2 milli-
meters in fork length were obtained from hatcheries from September 23,
1965 to March 22, 1966.
Atlantic salmon (Salmo salar), Lahontan cut-
throat (S. clarki henshawi), and kokanee, which are landlocked sockeye
salmon (Oncorh.ynchus nerka), were obtained from the Wizard Falls Hatch-
ery in Oregon; rainbow trout (S. gairdneri) from the Kiamath Hatchery
in Oregon (these fish were hatched from eggs obtained from brood stock
maintained at the Roaring River Hatchery in Oregon); Montana blackspotted cutthroat trout (S. clarki lewisi) from the U. S. National
Fish Hatchery at Creston, Montana; coastal cutthroat trout (. 2.
clarki) from the Cedar Creek Hatchery in Oregon; and coho salmon
(0. kisutch) from the LLsea River Salmon Hatchery in Oregon.
All
hatcheries except the last two arid the Roaring River Hatchery are
located outside the enzootic area or the "salmon poisoning" fluke.
Fish were obtained soon after hatching from the last two hatcheries
during the time of year when cercariae of
emerging from snails in streams.
.
salmincola are not
In all cases, 10 fish of each
species were examined by the squash method described below for
natural infection before others of the same species from the same
lot were used in an experiment.
Natural infections were not found
in any fish, but an infection was found in a coastal cutthroat trout;
it had two parasities.
This fish, a part of the control group in
Experiment 3, was apparently accidentally infected in the laboratory.
Stock fish were maintained in 620-gallon circular tanks of wood or in
plastic trays.
The tanks or trays were provided with flowing
chlorine-free tapwater at temperatures from 6 to 10°C and at pH from
7.0 to 7.8.
The pH was adjusted by bubbling carbon dioxide through
the water before it entered the tanks.
was determined daily.
The pH of the incoming water
Dissolved oxygen content, maximum and minimum
temperatures, and pH of the tank water were recorded weekly.
Oregon
moist pellets (small size) manufactured by Bioproducts in Warrenton,
Oregon, were fed to the fish once each day.
Collection of cercariae
In addition to N. saincola, 0. silicula can be parasitized by
at least fi'c.ve other species of digenetic trematodes (Burns, 1961), the
cercariae of which, however, do not penetrate fish (Bennington and
Pratt, 1960).
Mixed infections involving N. salrnincola are rare.
Bemüngton (1951) examined 100 snails and found no mixed infections.
I examined approximately 350 snails and found no mixed infections.
Gebhardt (1966) reported only 15 of 1695 snails to have mixed infections.
Only N. salmincola cercariae emerged from the snails in
my experiment during the entire study period.
salmincola are easily recognized.
The cercariae of
.
The identification, as
salmincola, of the cercarjae I used to infect fish was verified by
microscopic examination during the counting procedure described below.
Twelve hours before cercariae were needed for an experiment,
200 to 300 infected snails were removed from the trays and washed in
tapwater.
Each snail was then placed in a 60 x 20 millimeter Petri
10
dish with 15 milliliters of tapwater at 20 to 22°C under ceiling
light.
The pH of the water had been adjustedpreviously from ap-
proximately 10.0 to 7.0 to 8.0 by continuous aeration for 12 hours.
Light did not appear to stimulate emergence of cércariae, because in
preliminary experiments cercariae emerged equally well when the snail
is held either in darkness or approximately 12 inches from a fluorescent light.
Most of the crcariae began emerging 3 to Li hours after
isolation, and 25% to 30% of the snails produced large numbers of
cercariae during the 12-hour isolation period and only these cercariae
were used in an experiment.
Cercariae were pooled after 12 hours by
pouring the water from the Petri dishes into a 1,000 milliliter beaker.
Thirty minutes later the parasites were recovered from the bottom
of the beaker.
Infection of fish---morta1y studies
All of the fish species mentioned previously in the section
Fish procedures, except Lahontan cutthroat, were used in this phase
of the study.
Sixty to 90 fisri of one species were placed in a 5-
gallon jar containing 5 gallons of tapwater at 10°C.
The temperature
of the water was raised to 20°C by the addition every 10 minutes of
100 milliliters of boiling water during a 1 hour period.
was then placed into a separate
Each fish
ounce paraffin-coated paper cup
which contained 30 milliliters of pH-adjusted tapwater at 20°C and
at pH of 7.3 to 7.8.
exposure period.
The water was aerated continuously during the
Pooled cercariae were pipetted into Petri dishes so
that separate small drpps of water were formed.
The cercariae in
each drop were counted under lOX magnification.
A known number of
11
cercariae, 50 to 1,000, were then washed into the cups containing the
fish, and the fish exposed for 1 hour at 20°C which was a convenient
laboratory room temperature.
Ten fish were used for each exposure
level and 50 to 70 fish in each experiment.
Control fish were sub-
jected to the same conditions except that cercariae were removed from
the water to which the controls were exposed, by filtration through
No. 41 Whatman filter paper.
After exposure, the water from the cups
used for the exposure at each level was pooled and examined for cercariae.
Usually no more than 10 living and 20 to 30 dead cercariae
(the number depending on the exposure level) were found.
The fish in
each experiment were of the same age and approximately the same fork
length (Table I).
The length was obtained from fish that died during
the first 24 hours of the 30-day experiments.
Fish from each exposure level were pooled after exposure and
placed in 1-gallon glass jars containing 1 gallon of continuously
The dissolved
aerated tapwater in a 16°C constant temperature room.
oxygen, pH, and temperature of the water were determined when the
fish were placed in the jars and whenever fish died.
The pH values
ranged from 7.3 to 7.9, and the dissolved oxygen values ranged from
8.1 to 10.1 milligrams/liter.
The fish were fed Oregon moist pellets
daily approximately 1 hour before the water in each jar was changed.
Fish were observed for signs of infection every 6 to 8 hours during
the first 24 hours after exposure and thereafter.daily.
Deaths of
fish were recorded at these times, using cessation of respiratory
movements as the criterion of death.
The total number of encysted
metacercariae in experimental fish that died between 24 hours and
12
30 days or were killed after 30 days was determined by squashing the
fish between two 3 inch x 4 inch glass plates.
The plates, held to-
gether by four small "C" clamps, were placed over a grid under 15X
magnification and the parasites counted.
Control fish were similarly
examined.
The 24-hour mortality percentages were plotted against the exposure levels on semilogarithniic paper.
The 24-hour lethal exposure
level (24-hour LEL50--the exposure level, in numbers of parasites,
lethal to 50% of the fish in 24 hours) was determined for each species
by interpolation after fitting a sigmoid curve by eye to all of the
experimental data.
The average number of parasites in fish that died
between 24 hours and 30 days and in fish that were killed at the end
of 30 days following exposure to each tested level was plotted against
the 24-hour per cent mortality for the same exposure level.
The 24-
hour lethal dosage level (24-hour LD50--the infection level, in numbers
of parasites, that results in
o% mortalityof the fish in 24 hours)
was estimated from these curves for each species.
Parasites were not counted in fish that died sd-thin the first
24 hours.
During this time cercariae were not encysted and their
excretory bladders were not filled.
Thus, they were not clear±y
visible for counting by the squash technique0
They were also too
fragile to be counted by the homegenization-sedimentation technique
outlined in Gebhardt et al. (1966).
Therefore, I have assumed that
the number of parasites in fish that survived for the first 24 hours
approximates the number present in fish that died during this time.
13
Penetration and gtn of cercariaein fish;
1.
Percutaneous exposure
Initial penetration of cercariae into fish was studied by placing
cercariae on the caudal fin of intact pithed fish (Lahontan cutthroat
trout) and on 1 centimeter sections of caudal fin clipped from pithed
Lahontan cutthroat trout, and then observing penetration under 15X
magnification.
Subsequent migration of the parasites in fish and the
histopathologic changes associated with this migration were studied
by examining histological sections.
Each of six Lahontan cutthroat
trout, 42 to 60 millimeters in fork length, was exposed to 500 cercariae in the manner previously described.
each of the following times after exposure:
144O minutes.
tive.
One fish was killed at
15, 30, 60, 120, 720, and
The fish were fixed for 24 hours in Mossmants AFA fixa-
A cross-sectional cut was made through the body of each fish at
the anterior edge of the dorsal fin and a second cut was made 1 centimeter posterior to the first.
This block of tissue was mounted. in par-
afiin and sections were cut at 15 microns.
Fery. tenth section of the
ribbon was stained with Harris' alum hemator1in and eosin.
Twenty-
seven sections were examined from each exposed fish and 3 to 4
sections from a nonexposed control fish.
2.
Alimentary canal exposure
Each of ten Lahontan cutthroat trout 60 millimeters long and
each of eight rainbow trout 110 to 150 millimeters long was exposed
to 100 and 500 cercariae, respectively, by stomach tube, using a 2
millimeter inside diameter nylon tubing attached to a 2 milliliter
hypodermic syringe.
Two control cutthroat trout were used to test
14
for infectivity of the cercariae and were exposed percutaneously.
Another group of five rainbow trout 150 millimeters long was exposed
to unknown numbers of cercariae by stomach tube on three different
days during a period of 6 days.
Also, each of six Lahontan cutthroat
trout 60 to 70 millimeters long received by stomach tube two cubic
sections 2 to 3 millimeters on a side of infected snail ovarian tissue.
Ten control fish received non-infected ovarian tissue.
To test for infectivity of the parasites, 700 48-day-old metacercariae were recovered from fish that had received free cercariae by
stomach tube.
tube.
These were given to each of two hamsters by stomach
The animals were killed 14 days later and the small intestine
examined for adult flukes.
II.
Eiergence of N. salmincola cercariae from snails held in waters
of different salinities.
Snails were collected from brackish water at two locations on
the Yaquina River in western Oregon.
These sites, areas 2 and 3,
were 2.5 and 5 miles, respectively, downstream from Elk City, Oregon
(Figure 1).
High and low tide salinities at area 2 was 9.4 and 2.2 ppt
on September 8, 1965, and at area 3 was 20.5 and 4.1 ppt on August 25,
1965.
Between 150 and 200 snails were collected at area 2 and 300
snails were collected at area 3.
Snails infected with N. salmincola
were separated from those infected with other species of trematodes
and were maintained, in a plastic holding tray containing diluted sea-
water at 4 ppt salinity.
Infected snails were isolated individually in 60 x 20 millimeter
Petri dishes containing tap water that had the pH adjusted previously
Toled
Scale:
1 inch = 1 mile
Figure 1.
Map of part of the Yaquina River system drawn from the
United States Geodetic Survey map of the Toledo quadrangle.
circled nimibers designate snail collection sites.
The
The head of
tidewater is located at the junction of shaded and unshaded parts
of the river.
16
from approximately 10.0 to 7.0 to 8.0 by continuous aeration for 12
hours.
The freshwater was replaced with seawater diluted with pH-
adjusted tapwater to give a salinity of Lppt at the end of 24 hours.
At the end of each succeeding 24-hour period the seawater was replaced
The tested salinities
with seawater of the next highest test salinity.
4, 8, 12, 14, 16, 18, and 2Oppt prepared from seawater having
were:
a known salinity using pH--adjusted tapwater.
A 10 millimeter disc of
lettuce was placed in each dish for snaiLfood.
done in a 20°C room.
The experiments were
Cercariae were counted either directly or by
computing an average based on five 1.0 milliliter aliquots taken from
a homogenous suspension of cercariae.
III.
N.
Effect of different salinities on viability and infectivity of
aliuincola cercariae.
!ili
of cerc ariae
Infected snails obtained from freshwater (Figure 1, area 1)
and 10 snails from each of the brackish water areas (Figure 1) were
Isolated in individual 60 x 20 millimeter Petri dishes.
The former
were held in 15 milliliters of freshwater, and the latter in 15
milliliters of seawater at 10 ppt salinity.
every 4 hours for cercariae.
The dishes were examined
Cercariae, 4 hours old or less, from
snails froni each area were pooled and divided into
qual groups of
15, 20, or 25 in a drop of water in 60 x 20 millimeter Petri dishes.
Ten milliliters of freshwater or seawater of a given salinity, from 1
to 20 ppt, were added to each dish.
Salinities higher than 20 ppt
were not tested because this was the highest salinity recorded at collection site 3.
The dishes were examined immediately after the water
17
Cessa-
was added and thereafter every 4 to 7 hours for dead cercariae.
tion or movement and lack of response to vibration, tapping of the
dish, were used as criteria of death.
The experiments were terminated
when more than 50% of the cercariac were dead in each dish.
Infectivity of Cercariae
The cercariae used in these tests were those that emerged from
snails collected from the sites in brackish water.
The snails were
held in freshwater and in seawater at the different test salinities
described above.
Cercariae that emerged during each 24.hour period
from snails of one area were pooled and tested for infectivity by one
of three methods.
Cercariae that emerged in freshwater. were exposed
to living Lahontan cutthroat trout (45 to 50 millimeters in fork
length) in freshwater.
Approximately 400 cercariae from area 2 were
placed into each of three cups, each cup containing one fish.
These
fish were exposed percutaneously in the manner previously mentioned.
The second method involved those cercariae that emerged at 4 and 8 ppt
salinity.
These cercariae were exposed to a fish from brackish water,
the three-spine stickleback (Gasterosteus aculeatus microcephalus) obtained from Eckmann Lake, Oregon.
It was necessary to use this
salinity-tolerant fish because salmonids adapted to saltwater were
not available.
Gebhardt et al.. (1966) have shown that the stickle-
back can be experimentally infected with
j. salmincola.
The test
sticklebacks were assumed not to be naturally infected because examination of some from the same lot used in the experiment were negative
for N. salmincola metacercariae.
The third method involved those
cercariae emerging at 12, 1, 16, 18, and 20 ppt salinity.
These
parasites were exposed to pithed, intact Lahontan cutthroat trout in
a 90 x 15 millimeter Petri dish.
One fish was used for each test
salinity and for each snail collection area.
Penetration of the cer-
cariae into the caudal fin of the fish was observed microscopically.
I found that the stickleback was not as susceptible as salmonids to
the parasite.
Thus, it was necessary to use a pithed salmonid to
test for the infectivity of the small numbers of cercariae emerging
at the higher salinities.
IV.
Emergence of N. salmincola cercariae from the snail, Q. silicula.
A group of snails were placed in individual Petri dishes to
allow cercariae to emerge.
One dish in which large numbers of cer-
cariae were being produced was selected for examination and removed
to a new dish of fresh tapwater for observation.
The snail was held
dth fingers by the apical end in the dish of water with the aperture
upward and the apex to the left.
The snail was observed at 7x magni-
fication under a dissection microscope.
After the snail had extended
its head and foot to the bottom of the dish, a strong light was
directed into the mantle cavity for observation.
19
RESULTS
I.
Effects of N. salmineota cercariae on salmonid fishes under ex-
perimental conditions.
Infection of fish--mortality studies
Most of the experimental fish exposed to large numbers of cercariae died within the first 2Ll hours (Figure 2).
died during this time.
No control fish
Some experimental fish, but no control fish,
died 1 to 2 days after exposure.
Some experimental and control fish
of all species, except kokanee, died within 3 and 30 days after the
start of the experiments.. There was no apparent difference between
the numbers of experimental and control fish that died during this
time (Table I).
I feel that the results of coastal cutthroat trout Experiment
3 are more reliable than results from Experiments 1 and 2, because
64% of the cercariae penetrated and encysted in Experiment 3, and
only 43% in the other two experiments.
The former figure is within
the range (60% to 76%) of percentages recorded for the other species
of fish.
The use of defective cups in the first two experiments, in
which cercariae may have been trapped in the paper fibers, could have
been responsible for this difference.
The data, therefore, from
Experiments 1 and 2 are not shown in Figures 2 and 3, but are presented in Table I.
After exposure to 200 parasites, 100%, 90%, and 70% of the
kokanee, Montana black-spotted cutthroat trout, and Atlantic salmon,
respectively, died during the first 24 hours (Table I).
Only one
coho salmon and no rainbow trout or coastal cutthroat trout died
20
a
too,
a
0
a
0
a
90
0
40
0
S
3°
20o
4/
/
0
50
Figure 2.
600800
PARASITE EXPOSURE LEVELS
100
400
200
The 2Lhour mortalities of salmonid fish exposed to known
numbers of Nanophyetus salmincola cercariae, in relation to the ex-
posure levels.
The number beside each curve is the LEL50 value for
that species of fish.
Cutthroat Trout.
Mont B1Sp Cutth Trout
Coast Cutth Trout
Montana Black-Spotted
Coastal Cutthroat Trout.
Table I.
Results of experimental exposure of six species of saimonid fish to Nanophyetus saimincola.
Species
Age
(weeks)
Kokanee
Montana
black-spotted
cutthroat
trout
14
17
Length
(mm)
30
31
Used for
exposure
600
400
200
100
50
12
31
2
37
-
50-68
27-52
0
800
600
400
200
100
-
400
200
100
50
0
Coho
salmon
Recovered
Range
Mean
0
50
0
Atlantic
salmon
No. fish dead
No. parasites
Fish
800
600
400
200
100
50
0
58
10
10
10
39
2
0
5 6-79
77
67
35-44
38
0
-
0-i
-
145-162 154
66-84
77
32-43
37
0
275-335 305
101-154 122
5 0-98
72
36
30-55
0
5
10
10
10
9
2
1
0
10
7
2
0
0
10
10
8
1
0
0
0
Days after exposure
11-20 21-30
2
4-10
3
Totals
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ia/ia
10/ia
10/10
6/10
2/10
0
0
0
0
0
a
0
0
0
0
0
0
0
4
0
o
0
0
4
0
i
10/10
ia/ia
ia/ic
ia/ia
a
0
0
0
0
0
0
a
0
0
0
0
0
0
0
a
0
0
0
1
1
0
0
0
1
1
1
0
0
1
0
2
ia/ia
0
0
0
0
0
O
0
0
0
0
1
0
0
0
0
1
1
1
2
0
a
0
1
1
0
3
0
a
1
1
0
1
la/ia
ia/ia
9/ia
4/ia
3/10
2/10
9/10
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
00
00
4
o/o
6/9
Va
r//iO
s/ia
2/9
3/10
N)
Table I.
Fish
Species
Rainbow
trout
Coastal cutthroat trout
Exp. #1
No. parasites
Age
(weeks)
16
8
Length
(mm)
33
29
Used for
exposure
Coastal cutthroat trout
E3cp. #3
9
12
31
31
Recovered
Range
Mean
800
600
400
200
100
50
0
382
444
227-327 269
67-185 115
58
44-72
15-49
31
0
800
600
400
200
100
301-450 356
136-256 186
102-256 155
61-156 88
28-71
45
15-44
27
50
0
Coastal cutthroat trout
Exp. #2
(Continued)
-
0
387-559
196-384
109-344
107-291
42-142
0
495
298
219
800
600
400
200
100
0
422-531
323-373
203-378
108-177
48-88
467
348
276
138
68
180
95
Days after exposure
2
4-10
11-20 21-30
3
0-1
9
9
4
0
0
0
0
5
2
0
0
0
0
0
1,000
800
600
400
200
0
2
No. fish dead
0
0
0
0
0
0
0
0
00
0
0
0
2
0
2
0
0
0
0
0
0
0
1
2
3
2
2
2
0
0
1
1
0
0
0
0
0
0
01
0
0
0
0
0
0
0
0
6
0
0
4
0
0
1
0
0
0
1
0
0
0
0
7
7
2
1
0
0
0
0
0
0
00
0
0
0
1
Totals
0
0
0
2
1
1
2
9/10
9/10
5/10
4/10
4/10
4/10
3/9
5
4
4
0
0
1
1
0
1
1
0
8/10
6/10
7/10
5/9
7/10
7/10
6/10
0
0
3
4
0
1
1
0
1
1
2
1
0
3
0
1
0
0
8/10
10/10
6/10
7/10
2/9
2/10
1
1
4
2
4
0
0
0
0
9/10
10/10
9/10
8/9
8/10
10/10
5
5
4
0
3
0
1
1
1
3
3
5
3
2
5
1
0
N)
N)
23
100
j
0'
90
80
0I
cr1
70
I
Il
I-_I
I__I
>__60
DI
01
O58c?j74
/1/cciI
.I
0
40
25
Figure
/
0
u_I
50
60 80 100
PARASITE
DOSAGE
3.
n9p36
oI
0 /
fId
I295 J430
<>q'
(
200
300 400500
LEVELS
The 2k-hour mortalities of saonid fish infected
th
nC2a metacerCariae, in relation to estimated num-
bers of parasites.
The numbers (average) of parasit8S in these fish
were assumed to be the average numbers found in fish that were exposed to the same numbers of cercariae and that survived for the
first 2
hours of the experiments.
The number beside each curve is
the LD50 value for that species of fish.
Figure 2.
kbbreViations same as for
24
All of the coho
during this time at this exposure level (Figure 2).
salmon and 90% of the rainbow trout died within 24 hours after exposure to 600 parasites, whereas a 40% mortality (Experiment 3) of
the coastal cutthroat trout was observed (Figure 2).
The 24-hour
LEL505for kokanee, Montana black-spotted cutthroat trout, Atlantic
salmon, coho salmon, rainbow trout, and for coastal cutthroat trout
(Experiment 3) are 94, 135, 157, 315, 340, and 700, respectively
(Figure 2).
The highest parasite numbers in kokanee, Montana black-spotted
cutthroat trout, and Atlantic salmon that survived for the first 24
hours were 68, 79, and 162, respectively (Table 1).
One coastal
cutthroat trout that survived had 559 metacercariae, and seven had
between 400 and 550 parasites.
Coho salmon and rainbow trout that
survived had numbers of parasites less than those found in coastal
cutthroat trout but much greater than those found in the other named
species (Table 1).
The highest number of parasites in coho salmon
was 335, and one rainbow trout had 444 parasites.
The 24-hour LD50,5
for kokanee, Montana black-spotted cutthroat trout, Atlantic salmon,
coho salmon, rainbow trout, and for coastal cutthroat trout (Experi-
ment 3) are 58, 74, 110, 200, 295, and 430, respectively (Figure 3).
The average percentages and ranges of percentages (in parentheses) of cercariae that penetrated and encysted in each fish species
are as follows:
kokanee 68% (58 to 78%); Montana black-spotted cut-
throat trout 60% (38 to 76%); Atlantic salmon 76% (74 to 77%); coho
salmon 70% (61 to 76%); rainbow trout 61% (48 to 74%); coastal cutthroat trout 64% (58 to 69%).
These figures were calculated using
25
the numbers of parasites recovered from fish that survived for the
first 24 hours of the experiment.
Penetration and migration of cercariae in fish.
1.
Percutaneous exposure.
Cercariae penetrated the skin of the fish at any point and penetration was completed within 3 to .5 minutes.
These observations are
Uin agreement with those of Bennington and Pratt (1960), and contrary
to statements by Sinitsin (1930) and Sirnms, Donham and Shaw (1931), who
believed that the urinary aperture and gills were the portals of entr3l
I observed that cercariae became active when they were within 1 millimeter of the fishts skin.
Sktn penetration by the parasite involved
attachment of the ventral sucker and contraction of the body into an
egg-shaped mass.
The anterior end was then forced into the skin.
Cercariae migrated through the skin along a path parallel to the surface without the resting period mentioned by Bennington (1951).
Cer-
cariae that penetrated the caudal fin entered either blood vessels between rays, as first observed by Bennington and Pratt (1960), or blood
vessels inside rays (Figure 4).
fin.
They then migrated to the base of the
The circulatory system apparently is one of the principal routes
used by cercariae to reach internal organs, such as the liver and
heart, since parasites were seen adjacent to and inside blood vessels
(Figures 5, 6, 7) in the hypoderniis.
Cercariae were seen in renal
blood vessels; thus, they could reach the kidney, where they are
found in large numbers, via the renal portal system, as suggested by
Bennington and Pratt (1960).
Cercariae were also observed in the
peritoneum and body cavity and may enter internal organs from these
Figure 4.
Cross section through the caudal fin of a Lahontan
cutthroat trout, shovng two cercariae (C) inside a fin ray.
X 377.
27
b
-
Figure 5.
-'
1)
Cross section through the base of the caudal fin
of a Lahontan cutthroat trout 52 millimeters long exposed to
500 cercariae of N. salniincola, showing hemorrhagic area (H)
in loose connective tissue and a cercaria (C) near a fin ray
(F) and a blood vessel (B).
X 377.
Figure 6.
Cross section through the base of the caudal fin
of a Lahontan cutthroat trout, showing one or two cercariae
(C) of N. salmincola inside a blood vessel and two other
cercariae in the subcutaneous tissue.
X 377.
o
C
I'
I
LAii
Figure
7.
Higher magnification of Figure 6, showing
(C) in a blood
vessel.
X
1525.
cereariae
30
sites.
Many cercariae were seen in the body musculature.
These
parasites probably do not migrate to internal organs, but rather,
remain in the muscles, where they are found in large numbers.
Many
cercariae were found in the epidermis and hypodermis 15 and 30 minutes after exposure of fish, and by 60 minutes most of them were deep
in the body musculature and some were seen in the kidney for the first
time.
Symptomatic and pathologic changes
a.
Gradations of the following signs of infection were observed
in all infected fish but not in the controls.
A decrease in swimming
activity and an absence of the characteristic darting movement (except
for Atlantic salmon, which characteristically remain at the bottom of
an aquarium) appeared 1 to 2 hours after exposure.
Biting was not
common during the first 24 hours among infected fish.
Diffuse
petechiae on the body surface and larger hemorrhagic areas at the
bases of the pectoral, pelvic, and caudal fins were observed within
2 hours (cf. Figure 8 and 9).
Hemorrhagic areas reached a maximum
size 12 hours after exposure of fish and many were visible for 48
hours.
Equilibrium loss, drifting, erratic swimming, increased rate
of respiratory movements, and in some fish vertical or horizontal tail
curvature, were observed.
These changes were most pronounced in fish
that died within the first 24 hours, and were not always present in
surviving fish.
Papules were observed, on the skin of fish, pre-
sumably where cercariae penetrated.
Bennington and Pratt (1960).
dead fish.
These were first reported. by
Orbital hemorhage was seen in many
There were large numbers of extravasated red blood cells
I
a
, ..
,.. .I
I.
'I
I- :,,
/11
: \\\..
1
/11 1ii
Figure 8.
Photograph of caudal region of a Lahontan cutthroat
trout exposed to 700 cercariae of
ncola 21-J-hours
before the picture was taken, showing large hemorrhagic area.
X 22.
'4,,
'I_:
I
Figure 9.
\ui.
Caudal region of an uninfected Lahontan cutthroat
trout 45 millimeters long.
X 22.
33
in the loose connective tissue between the dermis and muscle bundles
in the hemorrhagic area at the caudal fin base (Figure 5).
The
hemorrhage probably was due to destruction of the blood vessels by
migrating cercariae.
Destruction of muscle tissue posterior to a
cercaria was observed and may have resulted from migration of the
parasite.
Partial renal tubule and blood vessel occlusion were ob-
served adjacent to a parasite.
This is the first detailed report on
pathologic changes associated with penetration and migration of N.
salniincola cercariae.
Wood and Yasutake (1956) studied the histo-
pathology associated with encysted metacercariae in naturally infected fish.
2.
Alimentary canal exposure
The majority of fish that received either infected snail
tissue or cercariae by stomach tube were infected.
However, the
number of xnetacercariae recovered from these fish was less than the
number recovered from the percutaneously-exposed control fish.
Para-
sites from the experimental fish were found in the heart, liver, kidney, intestinal wall, roof of the mouth, and gills.
These parasites
were infectious, as shown by recovery of adult flukes from hamsters
that received xnetacercariae 14 days previously.
The presence of
metacercariae in internal organs of the experimental fish suggests
that they may have developed from cercariae that reached these sites
via the circulatory system or by penetration of the alimentary tract
wall.
These suppositions are in agreement with observations on
histological sections of infected fish.
I believe on the basis of
the above findings that fish in nature may become infected
34
percutaneously and orally by ingestion of infected snails or free cercariae.
Ching (1957) and Gebhardt (unpublished observation) reported
the occurrence of snails in fish stomachs.
Gebhardt (unpublished) has
observed that coho salmon 46 millimeters long will become infected in
the laboratory after avidly eating freely falling cercariae.
II.
Emergence of N. salinincola cercariae from snails held in
waters of different salinities.
There were no deaths of snails held at the various test
salinities during the experimental period of
Cercariae
days.
emerged from snails from both brackish water areas at all of the
test salinities except those snails from area 3 held at
ppt salinity
(Table II).
There is an inverse relationship between increase in salinity
and the number of snails from which cercariae emerged.
Cercariae
emerged from only one snail of ten from each area at 20 ppt salinity,
and only a few cercariae emerged from these snails.
However, even
at relatively high salinities an appreciable number of cercariae
emerged, e. g. a total of 1,500 cercariae emerged from two snails
from area 2 at 16 ppt salinity.
The results of this study show that
cercariae can emerge from snails, and also that snails can live for
several days, at salinities higher than those that the snails encounter in nature.
III.
Effect of different salinities on viability and infectivity
of N. salmincola cercariae.
Viability of cercariae
Cercariae from snails collected from both freshwater and
35
Effect of different salinities on emergence and infectivity
Table II.
of N. salinincola cercariae.
Total no.
snails from
Salinity
Snail
source
Days
Area 2
1
2
(ppt )
0
4
which
cercariae
emerged of
10 initial
7
7
Total
cercariae
emerging
(aliquot
or actual
count
2,240
30,000
Method of
testing infectivity
Exposed
400 cerc.
to each of
3 fish (cutthroat).
Results
of
test
187
cysts
recovered
No cysts
osed all
recovered
cerc. to 1
fish (stickleback).
Area 3
1.
3
8
7
500
4
12
7
1,500
5
14
4
250
6
16
2
1,500
7
18
2
165
8
20
1
10
1
0
2
100
2
4
4
2,000
3
8
0
0
H
Exposed all
cero. to
pithed fish
(cutthrbat).
Observed
penetra-
Exposed all
cerc. to 1
fish (cutthroat).
2 cysts
recovered
tion.
Exposed all
No cysts
cerc. to 1
recovered
fish (stickleback).
Fish were examined for metacercariae by the homogenizationsedLmentation technique outlined in Gebhardt, et al. (1966)
(Continued)
'table II.
Total no.
snails from
Snail
source
Salinity
Days
(ppt )
which
cercariae
emerged of
10 initial
Total
cercariae
emerging
(aliquot
or actual
count
4
12
4
25
5
14
1
10
6
16
2
20
7
1
5
20
20
1
3
Method of
testing in-
fectinty
Exposed all
cerc. to
pithed fish
(cutthroat).
Results
of 1
test
Observed
penetration
37
brackish water survived the longest (75 hours) at saUnities between
Cercariae in freshwater had a shorter sur-
2 and 6 ppt (Figure 10).
vival time (38 hours).
At 12 ppt one-half of the cercariae from
snails collected from freshwater were alive after 4 hours, whereas,
one-half of the cercariae from snails collected from brackish water
were alive after 20.5, 26, and 38 hours.
Cercariae from area 3
snails survived the longest (38 hours) at 12 ppt.
At 16 ppt, one-
half of the cercariae from snails from areas 2 and 3 were alive at
5, 6, and 9 hours.
Cercariae from snails from freshwater survived
less than 5 hours at 13 ppt salinity.
It appears that at lower salinities (2 to 6 ppt), cercariae
from snails from the highest salinity collecting area did not do as
well as cercariae from snails collected from freshwater.
However,
further experiments must be done to prove this conclusively.
Infectivity of Cercariae
Cercariae penetrated fish at all test salinities except at
4and8ppt.
I have no explanation why cercariae did not penetrate fish at
these salinities, but did so at higher salinities.
A partial reason
may be that a more susceptible species of fish (cutthroat trout) was
used in the tests involving salinities greater than 4 pp; whereas,
the stickleback was used to test for infectivity of cercariae shed
at 4 and 8 ppt salinity.
I have assumed that cercariae that penetrated fish in these
experiments would have encysted; however, this was not determined
in these studies.
(1)
0::
0
=
w
I-
-J
>
U)
0
0
I
Z
V
I
V
V
I
3
4
5
6
7
V
8
V
9
I
10
V
II
I
12
I
I
13
14
I
15
I
V
16
Il
I
18
19
20
SALINITY (0/)
Figure 10. Survival times in hours of 50% of different groups of cercariae
placed in fresh water and different concentrations of sea water (ppt). Cercariae came from snails collected from area 1 (0), area 2.
(
and 0 ),
and area 3.
(L ).
39
IV.
Friergence of N. salinincola from the snail, Q. silicula.
Cercariae were carried rapidly in the blood inside the penvisceral, rectal, and mantle sinuses.
These sinues are connected
to the afferent ctenidial sinus at the right margin of the ctenidium
according to Ching (1957).
Cercaniae passed into the afferent
ctenidial sinus and then to the central sinus of each etenidial
leaflet.
At this time the cercariae suddenly stopped drifting.
They orientated lengthwise in the central sinus and moved by alternate
contraction and extension of the body to the ctenidial tip where they
emerged, anterior end first, into the mantle cavity.
The cercaniae
began to contract and extend rapidly, as soon as they were completely
free of' the etenidial tip.
They drifted out of the mantle cavity in
the exhalent current of water on the right side of' head (Figure ii).
Bennington and Pratt (1960) stated that cercariae were released from
snails in ropy strands of mucus.
I never observed this, but I did
find that cercariae after several hours became entangled in mucus
secreted by the snails if the latter were confined in small vessels.
No cercariae were ever seen emerging from the surface of the foot or
head.
However, histological sections revealed cercariae within the
foot muscle.
40
-M
Cs
CL
-
-_
->-_
_-:::_
Figure 11. Mantle cavity of the snail, Oxytrema silicula.
Central
AC = Afferent ctenidial sinus, CL = Ctenidial leaflet, CS
InRead, I
sinus, CT = Ctenidium, E = Exhalent water current, H
Mantle, NC = Mantle cavity, S = Shell.
halent, N
41
DISCUSSION
This is the first report on the pathogenicity of N. salmincola
for fish under controlled conditions involving known numbers of parasites,
Deaths of experinental fish within the first 24 hours after
exposure to cercariae are attributed to the effects of the parasites
because no controls died during this time.
Philip (1955) reported
that the etioLogic agent of "salmon poisoning" disease, Neorickettsia
he],ininthoeca did not affect trout fingerlings.
Thus, it is unlikely
that the rickettsiae could have caused the death of the fish.
Deaths
of fish after the first 2 days of the experiments could not be definitely attributed to the parasites because controls also died,
These
fish may have died because conditions were not satisfactory for their
maintenance in the laboratory.
For example, aggression (fighting and
biting), presumably a result of crowding, was common among the coho
salmon, rainbow trout, and coastal cutthroat trout, and many had
body and fin damage.
It is clear from my studies that there are large differences
in sensitivity to the early stage of infection among the different
fish species tested.
The five species of fish can be divided into
three groups having different relative sensitivities, as shown by
the LEL and LD curves (Figures 2 and 3).
trout is the most resistant species.
The coastal cutthroat
The rainbow trout and coho
salmon are intermediate in resistance, and kokanee, Atlantic salmon,
and Montana black-spotted cutthroat trout are the most sensitive
specLes.
The high virulence of the parasite for Montana black-
spotted cutthroat trout and Atlantic salmon may account in part for
42
the lack of success of efforts by the Oregon State Game Commission
to establish these species of fish in streams within the enzootic
area.
It is widely accepted principle that the pathogenicity of a
parasite usually will be reduced with increasrig duration of the
host-parasite relationship.
The results of my experiments appear to
be generally in accord with this principle, which, therefore, may
explain the differences in sensitivities of various salmonid fishes
to N. samincola.
Atlantic salmon, Montana black-spotted cutthroat
trout, kokanee, and their parent stock, sockeye salmon, except sockeye salmon in the Columbia River that are destined for streams east.
of the Cascade Mountains, do not occur naturally in the enzootic
area of the "salmon poisoning" fluke (Snyder, 1940; Oregon State
Game Commission, 1947; Carl, Clemens and Lindsey 1959).
species tested occur naturally in the enzootic area.
The other
Thus, Atlantic
salmon, Montana black-spotted cutthroat trout, and kokanee should be
the most sensitive of the tested species to the effects of the parasites.
The genus Oncojs is more recent in origin than SaLmo,
having evolved from the latter in the western Pacific Ocean (Neave,
l9).
c2hus presumably became established in the enzootic
area later than did Salino.
Coastal cutthroat trout, therefore,
should be more resistant to the parasite than coho salmon.
The
rainbow trout used in my studies were hatched from eggs obtained
from brood stock maintained at a hatchery within the enzootic area.
The history of this stock is not completely known, and therefore,
it is difficult to speculate as to the significance of the relative
43
resistance of the rainbow trout to the infection.
This rainbow
trout stock caine to Oregon from Utah approximately 30 years ago and
thus this species should be as sensitive to the effects of the parasite as the other exotic species tested; however, the rainbow trout
are more resistant than the non-native species of fish.
This may be
explained either as a result of selection or genetic contamination
of the stock by either intentional or accidental interbreeding with
trout native to the enzootic area.
It is known that the rainbow
trout stock tested was interbred with native rainbow trout approxixnately 25 years ago.
However,, factors other than previous contact
of rainbow trout with the parasite may be involved in determining
the relative resistance of this fish species.
Under the conditions of my experiments, the parasites killed
fish during the time of their penetration and migration.
The sur-
viving fish may be weakened physiologically, as suggested by Wood
and Yasutake (1956), during the later stages of infection when the
parasites are encysted.
Thus, the growth or swimming ability of
such fish may be impaired.
Whether fish in nature encounter during a short period of
time the numbers of cercariae that I used is not known.
It is
possible that fish can tolerate daily exposure to low numbers of
parasites over a period of time, as stated by Farrell and Lloyd
(1962), and thus accumulate large numbers of parasites that if
acquired within 24 hours would be lethal.
This would explain the
large numbers of parasites found by Simms, Donham and Shaw (1931)
and Simms et al (1931) in naturally infected fish.
It is also
44
likely that exophthalmia is a result of chronic exposure of fish to
low numbers of parasites, since I, in agreement with Bennington and
Pratt (1960) and Farrell and Lloyd (1962), did not observe this
condition.
The pathogenesis of the infection is not completely understood.
It is probable that N. salmincola cercariae secrete proteolytic
enzymes to aid in tissue migration, and high enzyme concentrations may
be toxic to fish.
The more resistant species of fish may have evolved
natural antibodies to these enzymes.
Such antibodies would be absent
in the most sensitive species of fish.
Gobhardt (1966) found 0. silicula snails infected with N.
in brackish water having a maximum salinity of 10ppt.
My
finding infected snails in brackish water having a maximum salinity of
20 ppt shows that this freshwater snail and N. salmincola have adapted
to or retained a tolerance for relatively high salinities.
The only
known published reports on the natural occurrence of other freshwater
mollusks in saline water are those of Karpevich (in Zhadiri, 1965) and
Segerstrle (1957).
Karpevich found species of the freshwater bivalve,
ssena in brackish water in the Caspian Sea.
the occurrence of the snails,
inaea
Segerstrle reported
tentaculata,
and Theodoxus fiuviatilis, and the bivalves Anodonta cygnea and
in the Baltic Sea.
This is the first reported study on the salinity tolerance of
cercariae from a freshwater snail.
Previous workers (Stunkard and
Shaw, 1931; Rees, 19Ll8; and Sindermann and Gibbs
cercariae from marine snails.
1953) have used
Stunkard and Shaw (1931) stated that
45
a study on the effects of diluted seawater on freshwater cercariae
was in progress at that time. The results of this study to my knowledge have not been published.
My results show that
sa1minc
cercariae can emerge from snails held in a salinity as high as 20 ppt.
A salinity at which cercariae stopped emerging was not determined;
however, it would probably not be much abcve 20 ppt because at this
salinity few cercariae emerged.
The optimum salinities for survival
of cercariae from snails from freshwater and from brackish water were
between 2 and 4 ppt, and 2 and 6 ppt, respectively.
Large numbers of
infected snails could not be obtained from brackish water and this
precluded a more precise determination of the optimum survival
salinity for cercariae from these snails.
The findings of Shaw
(unpublished) that N. salinincola cercariae live longer at low
salinities than in freshwater agree with mine.
cercariae survived (number not given) for 4
sodium chloride and tap water, respectively.
He reported that
and 41 hours in
ppt
I found that 50% of
the cercariae survived for 45 and 25 hours at 7ppt salinity and in
freshwater, respectively.
The reason why cercariae survived longer
in water having a low salinity than in freshwater may be explained
by the findings of Picken (1937) who worked with the snail, Limnaea
(sic) peregra.
He reported that the vapor pressure of the snail
blood was equivalent to a solution of sodium chloride at ca. 4.3ppt
salinity.
The results of my study suggest that N. salmincola cercariac
have adapted to or retained a tolerance for saline conditions because they are able to emerge, survive, and penetrate fish at a
46
salinity as high as 20 ppt.
The finding that cercariae from snails
from brackish water lived longer at salinities above 12 ppt than
cercariae from snails from freshwater suggests some physiological
adaptation by the parasite to saline conditions.
If fish can be infected in brackish water in nature, as I have
shown they can be in the laboratory, then possibly adult salmon that
stray into brackish water within the enzootic area could become infected.
If some of these fish spawned in streams outside the enzootic
area then the disease agent could be carried to definitive hosts in
new areas.
Another implication from the finding that fish can be
infected in saline water is that a lethal parasite dosage could be
established in young fish if they remained in brackish water for
extended periods of time, or in young fish reared in brackish inpoundments having infected snails.
47
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